International Journal of Biotechnology for Wellness Industries, 2012, 1, 67-84
67
Study of Corylus cornuta Twig Extracts: Antioxidant, Radical
Scavenging, Anti-Enzymatic Activities and Cytotoxicity
Mariana Royer and Tatjana Stevanovic*
Centre de recherche sur le bois, Département des sciences du bois et de la forêt, Pavillon Gene H. Kruger,
Université Laval, Québec, G1V0A6, Canada
Abstract: In a fist attempt to better understand therapeutic uses of the forest species Corylus cornuta by Native People
of Eastern Canada, antiradical/antioxidant, anti-enzymatic activities as well as cytotoxicity on Normal Human
Keratinocytes (NHK) of C.cornuta twig extracts were studied and correlated with their polyphenolic composition.
Polyphenolic extracts were obtained by water and ethanolic extractions using two different techniques: maceration and
ultrasound-assisted extraction. Antiradical and antioxidant capacities of the extracts were evaluated against DPPH,
TEAC, six ROS/RNS and peroxidation lipidic. Anti-enzymatic activities against enzymes involved in oxidation processes
were evaluated towards catalase and xanthine oxidase. MTT and Neutral Red assays were used for evaluating the
toxicity of the various extracts on NHK after 24 and 48h exposition times. Aqueous extracts were determined to have the
highest antioxidant/antiradical capacity against two reactive species involved in inflammatory processes (superoxide
anion and nitric oxide) and the lowest toxicity. Their antioxidant/antiradical activities were strongly correlated to their
higher content in flavonoids. Ethanolic extracts were determined to have the highest anti-enzymatic activity correlated
with their higher content in hydroxycinammic acids and proanthocyanidins. These extracts were also the most toxic, this
toxicity correlating with their high level in total phenols. Given that aqueous extracts presented an elevated content in
total phenols and flavonoids and showed the lowest toxicity on NHK as well as a high antiradical/antioxidant capacity,
they can be considered as the most valuable extracts obtained from C.cornuta twigs which is in harmony with traditional
uses in which remedies are prepared from twig infusions.
Keywords: Corylus cornuta, phenol contents, antioxidant/antiradical, enzymes, lipid peroxidation, cytotoxicity.
1. INTRODUCTION
Beaked Hazel (Corylus cornuta Marsh., Betulacea)
is the only hazelnut species growing in forest. Several
ethnobotanical and ethnopharmacological researches
showed that Native Americans used parts of beaked
hazel to treat various diseases [1-3]. For example, the
Iroquois prepared tea from the branches to treat dental
pain and they made necklaces out of pieces of stem to
ease tooth pain in young children. The Ojibwas used
the root mixed with other species to treat pulmonary
hemorrhage. The Algonquins and the Crees drank tea
from twigs, branches and leaves to treat heart disease
and intestinal disorders. The Potawatomi used the
inner bark in medicinal combinations in very much the
same way as they used the inner bark of the willow,
and also as an astringent and febrifuge [4]. The
Abenakis infused the bark with that of American
dogwood and willow to treat eye problems. There are
numerous testimonies today from people using beaked
hazel necklaces or products based on beaked hazel
extracts about their positive effects on several
pathologies such as arthritis, gastric ulcers,
cardiovascular disorders, teeth pains and skin
disorders (eczema, psoriasis).
*Address corresponding to this author at the Centre de recherche sur le bois,
Département des sciences du bois et de la forêt, Pavillon Gene H. Kruger,
Université Laval, Québec, G1V0A6, Canada; Tel: (418)656-2131 ext. 7337;
Fax: (418)656-2091; E-mail: [email protected]
ISSN: 1927-3037/12
Several studies have already shown the role of
oxidative stress in the mechanisms of various human
diseases such as cardiovascular [5, 6], lung [7, 8],
gastric ulcers [9-12] or even arthritic problems [13, 14].
In normal conditions, our body constantly produces
oxidant molecules that are highly reactive oxygenderived forms the function of which is to provide
signaling between cells [15]. The production of primary
reactive oxygen species (ROS) such as superoxide
anion (O2 •), hydroxyl radical (•OH) and hydrogen
peroxide (H2O2) is regulated by the body by an
enzymatic antioxidant system and also by the intake of
vitamins related to the daily diet. However, in some
cases (UV exposure, stress, alcohol, tobacco,
exposure to chemicals), there is an overproduction of
ROS. The antioxidant system becomes in that case
inefficient and an imbalance is created which is called
"oxidative stress". During oxidative stress, the ROS not
eliminated by the primary defense system of our body
will then oxidize cellular constituents which causes the
formation of secondary ROS (such as peroxyl radical
ROO•) that are participating oxidation chain reactions
which ultimately lead to cell destruction. The oxidative
stress is associated with lipid and protein peroxidation,
resulting in rapid cell structural damage, tissue injury or
gene mutation which ultimately lead to the
development of various health disorders such as
Alzheimer’s disease, cancer, atherosclerosis, diabetes
mellitus, and hypertension and finally ageing itself [16].
Under conditions of oxidative stress, excessive
© 2012 Lifescience Global
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International Journal of Biotechnology for Wellness Industries, 2012 Vol. 1, No. 1
Royer and Stevanovic
generation of •NO contributes to the inflammatory
response, in part by reacting with O2 • to form a potent
oxidant, peroxynitrite (ONOO–) / peroxynitric acid
(ONOOH). Phagocytes naturally produce oxidants such
as nitric oxide (•NO), superoxide anion, hydrogen
peroxide and hypochlorite ion (OCl ) as protective
agents against cell infection in immune responses [17].
Chronic infections involve a prolonged activity of
phagocytes, leading to inflammation and risks
associated with it [18]. It is well-known that chronic
inflammation is associated with a broad spectrum of
neurodegenerative diseases related to aging, such as
Alzheimer, Parkinson, amyotrophic lateral sclerosis,
age-related macular degeneration, osteoarthritis,
rheumatoid arthritis, atherosclerosis, and myocardial
infarction [19]. leaf). Indeed, various extracts obtained from hazelnut
by-products show antioxidant capacities including the
capacity to scavenge DPPH radical and to inhibit lipid
peroxidation [26-31]. Analyses of the chemical
composition of some hazelnut by-products do indicate
the high concentrations of polyphenolic compounds in
these extracts [32].
A therapeutic action aimed at correcting oxidant–
antioxidant imbalance is a clinical challenge, and has
increasingly been the object of research work.
Oxidative stress has been found to be controlled by the
antioxidant and/or anti-inflammatory effects of dietary
polyphenols. Since a variety of oxidants, free radicals
and aldehydes are implicated in the pathogenesis of
chronic
inflammatory
diseases, a
therapeutic
intervention with a variety of polyphenolic antioxidants
may therefore be an effective alternative for the
treatment of chronic inflammatory diseases [20].
Indeed, the role of polyphenols has been widely
demonstrated [21] and there have been new
developments in the elucidation of the in vivo
mechanisms of the health benefits of these compounds
[22].
2. MATERIALS AND METHODS
A very few phytochemical or pharmacological
studies of Corylus cornuta extracts are found in the
literature. McCune and Johns [23, 24] demonstrated
the high antiradical capacity of Corylus cornuta twig
and bark extracts, in particular against superoxide and
peroxyl radicals. These authors proved that Corylus
cornuta extracts showed the greatest antiradical activity
among 35 medicinal plants used by Indigenous People
in the boreal forest of Eastern Canada to treat diabetes
symptoms. Corylus cornuta were used in particular, to
treat symptoms such as diarrhea, heart/chest pain, and
sore eyes. However, several researches have been
conducted on the European species Corylus avelanna.
Thus, the tannin fraction of C. avellanna bark extracts
exhibits antibacterial activity against various strains of
bacteria [25]. It can be noticed that most of the
phytochemical studies of C. avellanna were performed
on fruits, leaves, oils or hazelnut kernel and on the byproducts (skin, hard shell, green leafy cover, and tree
The aim of this study was to better understand the
therapeutic uses of C.cornuta parts and extracts by
Native People of Eastern Canada. For the first time, the
composition of C.cornuta extracts in terms of various
classes of polyphenols was investigated and the
correlations between polyphenol content and
antiradical/antioxidant, anti-enzymatic activities as well
as cytotoxicity were investigated.
2.1. Plant Material
Plant material used in this study was harvested in
July 2010, identified and certified by Pure Hazelwood
Inc., Sherbrooke, QC. Since 11 years, this company
makes necklaces with C.cornuta twigs and proposes
skin products based on C. cornuta crude extracts.
2.2. Extractions
Water and ethanol were used as solvent for
extractions. With each solvent, two different extracts
were prepared, first by maceration, a classical
technique of extraction and the second one using a
modern technique of extraction assisted by
ultrasounds. Thus, the extracts of C.cornuta twigs
obtained by maceration in hot water (W Mac), by
extraction assisted by ultrasounds in water (WUAE), by
maceration in ethanol (EthMac) and by extraction
assisted by ultrasounds in ethanol (EthUAE) were
analyzed and tested in vitro.
2.3. Composition in Polyphenols
2.3.1. Determination of Total Phenol Content
The total phenol (TP) content of the extracts was
quantified according to Folin-Ciocalteu’s method as
described by Scalbert et al. [33]. 0.5 mL of sample
solution at concentration of 250 µg dry extract/mL was
mixed with 2.5 mL of the Folin-Ciocalteu reagent
(diluted 10 times by distilled water) and 2.0 mL of an
aqueous sodium carbonate solution (75 mg/mL). The
final mixture was heated at 50°C during 10 min, after
which the absorbance was read at 760 nm against a
blank (solution with no extract added). Gallic acid was
Study of Corylus cornuta Twig Extracts
International Journal of Biotechnology for Wellness Industries, 2012 Vol. 1, No. 1
used as the standard and TP content was expressed
as milligrams of gallic acid (Sigma-Chemical, St. Louis,
MO, USA) equivalents (GAE) per gram of dry extract
samples (mg GAE/g of dry extract).
2.3.2. Determination of Total Flavonoid Content
The total flavonoid (TFlav) content of crude extracts
was determined spectrophotometrically following the
method described by Brighente et al. [34]. Results are
expressed as milligrams of quercetin (Sigma-Chemical,
St. Louis, MO, USA) equivalents (QE) per gram of dry
extract (mg QE/g of dry extract).
2.3.3. Determination of Total Hydroxycinnamic Acid
Content
Total hydroxycinnamic acid (THCA) content was
determined as described in European Pharmacopoeia
th
(4 ed, [35]) for Fraxini folium. Total hydroxycinnamic
acid content was expressed as milligrams of
chlorogenic acid (Sigma-Aldrich, St. Louis, MO, USA)
equivalents (ChAE) per gram of dry extract (mg
ChAE/g of dry extract).
2.3.4. Determination of Proanthocyanidin Content
Anthocyanidin formation in a hydrochloric medium
with ferric ammonium sulphate (Laboratoire MAT,
Québec, Qc, Canada) as a catalyst was performed as
described by Porter et al. [36]. The proanthocyanidin
content (PAs) was expressed as milligrams of cyanidin
chloride (Indofine Chemical Co, Hillsborough, NJ, USA)
equivalents (CChE) per gram of dry extract (mg
CChE/g of dry extract).
2.4. Trolox Equivalent Antioxidant Capacity
The Trolox Equivalent Antioxidant Capacity (TEAC)
of C.cornuta crude extracts were evaluated by the
method of Prieto et al. [37]. This assay is based on the
reduction of Mo (VI) to Mo (V) by the sample and the
subsequent formation of a green phosphate/Mo (V)
complex at acidic pH. A total of 0.3 mL of methanolic
sample solution was mixed with 2.7 mL of the reagent
solution (0.6 M sulphuric acid, 28 mM sodium
phosphate and 4 mM ammonium molybdate). The four
C.cornuta twig extracts were prepared in order to be
analyzed at the same concentration in Total Phenols
(TP). Extract powders were dissolved in solvent to
reach 250 µg in TP expressed in gallic acid
equivalent/mL of extract solution. For the blank, 0.3 mL
methanol was mixed with 2.7 mL of the reagent. The
absorbance of the test sample was measured at 695
nm. Trolox was used as a reference and TAC results
69
were expressed as Trolox equivalents (mg TE/g of dry
sample). Extracts TAC were compared to that of the
®
standardized extract Oligopin prepared at 250 µg/mL.
2.5. Scavenging Capacity Against 2,2-diphenyl-1picrylhydrazyl (DPPH) Radical
-4
Methanolic solutions of 2.10
M DPPH· and
phenolic extract solutions were mixed in the ‘‘Rapid
Kinetics Accessory” SFA-11 (HI-TECH Scientific,
SALISBURY, England) and the absorbance of DPPH·
at 516 nm was monitored. The rate constant of the
hydrogen atom transfer pseudo-first order reaction, k,
was estimated using kinetic equation as described by
Diouf et al. [38]. Higher rate constant values
correspond to higher antioxidant capacities. All extracts
were prepared to reach 250 µg in TP expressed in
gallic acid equivalent/mL of extract solution and
compared to well-known antioxidants: ascorbic acid,
BHT, α-tocophérol and propyl gallate (Sigma-Aldrich,
Oakville, ON, Canada) used as positive references at
the concentration of 250 µg/mL.
2.6. Evaluation of Scavenging Activity Against
Reactive Oxygen (ROS)/Nitrogen (RNS) Species
2.6.1. In Vitro Tests, General Remarks
All ROS/RNS scavenging capacities studied in this
research were evaluated spectrophotometrically. A
commercial pine bark extract, which is a standardized
proanthocyanidin-rich extract from French maritime
®
pine bark (PBE), Oligopin obtained from DRT
nutraceuticals (DRT Nutraceutics, Dax, France) and
recognized for its several biological activities, was
chosen as the positive reference, like in our previous
study [39]. Absorbance was measured against a blank
solution that contained C.cornuta extracts or standard,
but without the reagent. A control was performed
without adding the Corylus cornuta extract or the
standard. The percentage of inhibition was calculated
by using the following formula:
%Scavenging = 100* (Acontrol-(Asample-Aextract))/Acontrol
where Acontrol is the absorbance of the control
(without antioxidant), Asample is the absorbance of the
solutions in presence of antioxidant and Aextract is the
absorbance of the blank (without reagent). The IC50
value, which is the concentration of the sample
required to scavenge 50% of reactive species, was
determined graphically from the curve plotted for
Corylus cornuta extracts or standards concentration
(µg/mL) vs. % inhibition. For comparison purposes, the
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International Journal of Biotechnology for Wellness Industries, 2012 Vol. 1, No. 1
antioxidant efficiency (AE) was defined and plotted as
the reciprocal of IC50 (1/IC50) so that the higher AE
value would correspond to better scavenging activity.
2.6.2. Superoxide Radical
Superoxide anion scavenging activity was
measured according to the method described by
Nishikimi et al. [40] with slight modifications. To 1 mL of
extract solution (0.01 to 1.0 mg/mL), 1 mL of 0.48 mM
β-nicotinamide adenine dinucleotide (NADH) and 1 mL
of 0.10 mM Nitro blue tetrazolium (NBT) prepared in
0.01 M phosphate buffer (pH= 7.4) were added. To
initiate the reaction, 100 µL of a fresh solution of 60 µM
of phenazine methosulphate (PMS) prepared in 0.1 M
phosphate buffer was added. After standing for 15 min
at 30°C, absorbance was read at 560 nm.
2.6.3.Hydrogen Peroxide
The procedure was adapted from Parij and Nève
[41] and performed at 25°C. To 100 µL of extract
solution (0.01 to 0.5 mg/mL) were added 2.7 mL of 0.1
M phosphate buffer (pH 7.4), 50 µL of a guaiacol
solution at 18 mM and 50 µL of hydrogen peroxide at
500 µM (Sigma-Aldrich, St. Louis, MO, USA). The
mixture was allowed to stand during at least 30 min.
Then the reaction was initiated by the addition of 100
-3
µL of a fresh horseradish peroxidase solution at 2.10
-9
mg/mL (~ 1.5 × 10 M). Absorbance was read at
436 nm.
2.6.4. Hydroxyl Radical
Hydroxyl radical scavenging activity was measured
as described by Smirnoff and Cumbes [42] with slight
modification. To 2 mL extract solution (0.01 to 1.0
mg/mL), 600 µL of 8 mM FeSO4 solution, and 500 µL
of hydrogen peroxide of 20 mM were mixed. Then, to
initiate the reaction, a 3 mM salicylic acid solution was
added. The reaction mixture was allowed to stand
during 30 min in a bath at 37°C, after which, 900 µL of
distilled water were added and the mixture centrifuged
during 10 min at 10000 rpm. The supernatant was
collected and the absorbance at 510 nm was recorded.
2.6.5. Peroxyl Radical
Peroxyl radical scavenging activity was determined
by the method described by López-Alarcón and Lissi
[43]. First, 300 µL of the extract solution (0.01 to 1.0
mg/mL) were mixed with 3 mL of a 30 µM of pyrogallol
red (PGR, Sigma-Aldrich, St. Louis, MO, USA) solution.
Then, 50 µL of a 600 mM AAPH 2,2’-azo-bis-2amidinopropane hydrochloride (Sigma-Aldrich, St.
Louis, MO, USA) solution was added to initiate the
Royer and Stevanovic
reaction. The samples were allowed to stand during 2 h
at 37°C. The absorbance was read at 540 nm.
2.6.6. Nitric Oxide
Radical scavenging of nitric oxide (NO) was
determined by the method described by Sreejayan and
Rao [44] with slight modifications. 200 µL of sodium
nitroprusside (SNP, Sigma-Aldrich, Oakville, ON,
Canada) (25 mM) was incubated with 800 µL of
C.cornuta extracts (0.01 to 1 mg/mL) dissolved in
phosphate-buffered saline (PBS) (25 mM, pH 7.4) and
the tubes were incubated at 37 °C for 2.5 h under
normal light exposure. After incubation, the samples
were kept in dark at room temperature for 20 min.
Thereafter, 300 µl of Griess reagent (Fluka,
Ronkonkoma, NY, USA) was added and the
absorbance was recorded at 546 nm after 40 min.
2.6.7. Hypochlorous Acid
Hypochlorous acid scavenging activity was
determined by the method described by Aruoma and
Halliwell [45]. HOCl was produced immediately before
use by adjusting NaOCl (1% v/v) to pH 6.2 with dilute
H2SO4 (25 mM), and its concentration was determined
as described by Wasil et al. [46]. Each extract or
reference solution (0.01 to 1.0 mg/mL) was mixed with
13 mM HOCl (4.3 mM, final concentration) in PBS at
pH 7.1 and incubated at 25°C for 10 min, followed by
the addition of 16.6 µM bovine liver catalase in PBS.
After 15 min incubation at 37°C, the absorbance was
read at 404 nm.
2.7. Capacity of the Extracts to Inhibit Lipidic
Peroxidation
The capacity of extracts to inhibit lipid peroxidation
was evaluated according to the method of Kuo et al.
[47]. The method consists of measuring the
peroxidation of linoleic acid in the presence of oxygen
catalyzed by hemoglobin. The results were obtained for
hazel twigs extract at phenol content varying from 0.1
to 1 mg / ml. The activity of the extracts was compared
to that of four standard antioxidants determined
previously: ascorbic acid, BHT, Trolox and propyl
gallate.
2.8. Anti-Enzymatic Activities
Due to their antioxidant activity, polyphenolic
compounds, by donating electrons, could change the
redox state of enzymes, particularly those that contain
metals as their prosthetic groups or react with free
radicals generated at the active site of the enzyme,
Study of Corylus cornuta Twig Extracts
International Journal of Biotechnology for Wellness Industries, 2012 Vol. 1, No. 1
thereby affecting enzyme activities [48]. The effect of
C.cornuta twig extracts on two enzymes that 1)
catalyze the oxidation of their substrate by molecular
oxygen and hence generate either intermediate or end
product-reactive oxygen species 2) limit oxidation
process by scavenging reactive oxygen species was
investigated. Two enzymes involved in antioxidant
processes were examined: one required to combat
reactive oxygen species that cause damage to cells
(catalase) and the other stimulating the production of
ROS (xanthine oxidase)
2.8.1. Xanthine Oxidase
The capacity of the extracts to inhibit xanthine
oxidase enzyme was determined by method described
by Cos et al. [49].The formation of uric acid is
measured spectrophotometrically at 290 nm. The
xanthine oxidase (Sigma Aldrich, EC 1.17.3.2.)
catalyses the formation of uric acid and superoxide
radical towards the reaction between xanthine and
oxygen. The extracts containing total phenols
concentrations ranging between 0.01 and 1 mg/mL
were tested. The activity of the extracts were compared
®
to those of the Oligopin , quercetin and curcumin
(Sigma Aldrich).
2.8.2. Catalase
The effect of the extracts on the catalase (Sigma
Aldrich, EC 1.11.1.6) an anti-oxidant enzyme was
deduced and discussed from the results of the in vitro
test performed with HOCl (2.6.7). Finding out that
certain extracts, beyond certain concentrations, had
tendency to favour catalase degradation instead to
inhibit it, our aim was to quantify the dose beyond
which the extracts started to have an antagonist effect
against this antioxidant enzyme, becoming “prooxidant”. The bovine liver catalase was used in this
study.
2.9. Toxicological Evaluation of Corylus cornuta
Extracts on Normal Human Kératinocytes
2.9.1. General Remarks
Extracts dissolved in culture medium were filtered
on 0.22 µm Millex GV filters (Millipore, Nepean,
Canada) and exposed to keratinocytes during 24 and
48 hours. The main outcome criterion used for
evaluating the toxicity of extracts was the determination
of toxicity value (IC50). IC50 represents the minimal toxic
dose required to cause the decrease of cell viability by
50 % compared with the untreated cells, which were
set to represent 100 % of viability. This value was
71
estimated from a dose-response curve, using a nonlinear regression algorithm [50]. Lower IC50 values
indicated higher toxicity.
2.9.2. Cell Culture
Normal human keratinocytes obtained from a breast
reduction surgery at passage 1 were cultured (6,7 x
2
103 cells/cm ) in the presence of a lethally irradiated
2
3T3 feeder layer (2,0 x 104 cells/cm ) in a combination
of Dulbecco-Vogt modification of Eagle's medium
(DMEM) with Ham's F12 (3:1) supplemented with 5%
Fetal Clone II serum (Hyclone, Scarborough, Ontario,
Canada), 5 µg/mL insulin (Sigma-Aldrich, Oakville, ON,
Canada), 0.4 µg/mL hydrocortisone (Cedarlane,
−10
Hornby, Ontario, Canada), 10 M cholera toxin (ICN
Biochemical, Montréal, Québec, Canada), 10 ng/mL
human epidermal growth factor (EGF) (Austral
Biological, San Ramon, CA), 100 UI/mL penicillin
(Sigma) and 25 µg/mL gentamicin (Schering, PointeClaire, QC, Canada). All cultures were incubated at
37 °C in an 8% CO2 air atmosphere and changed three
times a week with the media previously described. At
80 % confluence, keratinocytes were subsequently
subcultured after differential dissociation of the feeder
layers with 0.05 % trypsin-0.01 % ethylenediamine
tetraacetic acid (EDTA) treatment for 5 min. Cells at
passage 3 were used in this study.
2.9.3. Neutral Red Test
The cytotoxicity neutral red test is based on the
ability of live cells to uptake and bind neutral red (NR).
NR is a positively charged dye that easily diffuses
through the cellular membrane of the cells,
accumulates in the cellular cytoplasma and is stored in
the acidic environment of lysosomes. The principle of
the test is based on the fact that NR can only be
adsorbed by and bound to live cells, while this ability
declines in damaged or dead cells. The amount of
accumulated NR is thus directly proportional to the
amount of live cells in the cell culture. It is one of the
most used cytotoxicity tests with many biomedical and
environmental applications. The study was performed
on three cultures of normal epithelial keratinocytes of
adult humans (NHEK). Concentrations (1000, 500, 250,
125, 62.5, 31.25 and 15.625 mg / mL) of each product
were tested replicas (6). The confluence of NHEK at
the time of exposure was between 30 and 50%. After a
48 hours exposure, viability was measured by using the
neutral red (Neutral Red). A positive control (sodium
lauryl sulfate, SDS) was included in each experiment.
The results are expressed as IC50, which corresponds
to the dose of the product at which there is 50% cell
72
International Journal of Biotechnology for Wellness Industries, 2012 Vol. 1, No. 1
viability. The IC50 was calculated from a nonlinear
equation (Hill model) using the software GraphPad
Prism version 5.00.
This test was performed on cell cultures. In most
applications, the cells are placed in culture plates with
96 wells 6.4 mm in diameter. Each well can only be
used for a single determination. This experimental
device
allows
testing
of
multiple
chemical
concentrations of samples in duplicate, with positive
and negative controls. After the treatment of cells with
chemical products of concentrations ranging between
at least two orders of magnitude (for example, between
0,01 mM and 1 mM) and by chemical products
representing positive and negative control, the cells are
rinsed and treated with Neutral Red dye. Only the
viable cells are capable to incorporate and bind the
dye. The dye can either be added immediately after the
elimination of the tested chemical product, thus
determining its immediate effect, or it can be added at
variable intervals after the product elimination, in order
to determine its cumulative or deferred effect. The
colour intensity corresponds to number of viable cells in
each well. To measure this a spectrophotometer
equipped with plate reader programmed to measure
the intensity of each of 96 wells of culture plate is used.
This automated method yields quickly the results of the
concentration-response study and of the statistical
analysis.
2.9.4. Test with MTT
Another toxicity test is the relatively simple method
with MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole) which is reduced
to blue formazan in living cells. The MTT is a tetrazolim
type of dye which is reduced by mitochondrial enzymes
of the viable cells only. Therefore, the color intensity
measured is directly proportional to the degree of
integrity of mitochondria. This test is convenient for
detection of cytotoxic compounds in general but also as
the test for detection of agents attacking specifically the
mitochondria. Normal human keratinocytes were plated
4
2
4
into 96-well plates at 1.1 x 10 cells/cm with 2.0 x 10
2
cells/cm of 3T3 feeders for 3 days until 70 %
confluent. Another series of feeder cells culture also
4
2
seeded at 2,0 x 10 cells/cm were used to further
evaluate the contribution of irradiated cells to the
formation of formazan . After 24 and 48 hours of
exposure to the extracts and culture media, cells were
washed with phosphate buffered saline (PBS) and the
solution of methyl tetrazolium MTT (Sigma) (1mg/ml)
was added to each well during 2 hours. Then, the
Royer and Stevanovic
culture medium (0.2 ml) was aspired and replaced with
an equal volume of acidic Isopropanol (EMD
Chemicals) to dissolve the intracellular dark-blue
formazan crystal formed. After a few minutes at room
temperature, absorbance was read at 570 nm. The
cytotoxicity to keratinocytes was then determined by
subtracting the cell viability measured in keratinocyte +
feeder culture with that in feeder-alone culture as
described by Poon and Burd [51].
2.10. Statistical Analysis
The results are expressed as averages of three
measurements ± standard deviation. The ANOVA
variance analysis was applied to compare the averages
by Duncan test. The statistical significance threshold
was set at p < 0.05. The data were collected using
software R-Gui 2.112 http://www.R-project.org. The
non-parametric Spearman's correlation test was used
for correlations (p<0.05).
3. RESULTS AND DISCUSSION
3.1. Extraction
Composition
Yields
a nd
Poly phe nolic
The extraction yields and the chemical composition
of crude extracts from Corylus cornuta twigs are
presented in Table 1. The results show that water
extraction assisted by ultrasounds (W UAE) is less
effective than other extraction types in terms of
extraction yield. The extraction by maceration in hot
water is, on the contrary, the most effective in terms of
extraction yield. It is well-known that extraction with hot
water is a good method to recover polyphenols due to
their hydrophilic functions and a higher mass transfer at
high water temperatures [33]. Unlike aqueous
extractions, the ethanol, extraction yields are similar
regardless of the technique In the case of ethanolic
extractions; ultrasounds permit to reach the maximum
yield in a shorter time. Indeed, in preliminary
experiments, optimization of ultrasonic extractions was
done by following total phenol content (TP) in extract
solutions (ethanol or water) prepared with the same
initial mass of C. cornuta twig dry powder, the same
ratio of solid/liquid and in a temperature controlled
environment (data not shown). Lavoie et al. [52]
showed that optimization of extraction assisted by
ultrasounds can lead to the recovering of a great
amount of extractives in a shorter time and also can
permit to select bioactive compounds. Ultrasound is
known to facilitate the extraction of low molecular
weight molecules. It was reported that ultrasound
promotes the extraction of bioactive polyphenols [53].
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International Journal of Biotechnology for Wellness Industries, 2012 Vol. 1, No. 1
73
Table 1: Extraction Yields, Total Phenol, Total Flavonoids, Total Hydroxycinnamic Acids and Proanthocyanidins
Contents Determining Chemical Composition of Corylus cornuta Twig Extracts Obtained by Water and
Ethanolic Extractions
Extracts
WMAC
WUAE
EthUAE
®
Total Flav
TP
(%)
(mgGAE/ g dry
extract)
10.29
325.0 ± 9.1
7.69
EthMAC
Oligopin
Yields*
d
e
174.9 ± 1.1
c
9.84
410.7 ±14.5
9.96
b
-
433.9 ± 7.2
a
572.9 ±12.1
(mg QE / g
dry extract)
15.2 ± 0.6
a
7.2 ± 1.2
b
2.1 ± 0.2
d
4.3 ± 0.6
c
7.4 ± 0.1
b
Total CinnAc
% **flav
(mg ChAE/ g
dry extract)
4.7 ± 0.5
8.7 ± 0.7
4.1 ± 3.8
0.5 ± 0.1
1.0 ± 0.3
-
d
12.6 ± 0.4
d
44.6 ± 4.0
b
c
18.8 ± 2.6
a
335.5 ± 3.3
PAs content
%**
CinnAc
(mg CChE/ g
dry extract)
2.7± 0.2
10.5 ± 0.6
7.2 ± 0.2
10.9 ± 1.0
4.3 ± 0.6
-
%**PAs
e
3.2 ± 0.2
c
4.7 ± 0.2
b
6.6 ± 0.1
d
3.8 ± 0.1
a
-
15.1± 0.6
21.5 ± 0.2
12. 4 ± 0.3
105.0 ± 9.9
Means with different letters in the same column are significantly different at p<0.05 (ANOVA, followed by Duncan test). TP = total phenols content; Total Flav = total
flavonoid content; Total CinnAc = total hydroxycinnamic acids content, PAs content = proanthocyanidin content. WMAC = extract obtained by maceration in hot water;
WUAE = extract obtained by extraction assisted by ultrasounds in water; EthMAC = extract obtained by maceration in ethanol (at ambient temperature); EthUAE = extract
obtained extraction assisted by ultrasounds in ethanol.
* % (dry wt plant material) ** % correspond to the proportion of the polyphenol class reported to the total Phenol content of the considered extract.
Thus, depending on the solvent used, ultrasonic
extraction appears as a good alternative extraction
technique compared to conventional methods because
of its high efficacy, lower energy consumption and
lower water consumption (no need for refrigeration
because no reflux). The extraction time remains the
most important parameter that marks the difference
between the conventional techniques and ultrasoundassisted technique [54]. The effectiveness of this
technique has also been demonstrated in several
recent studies [55, 56].
Results in Table 1 confirm the preliminary
observations. Indeed, TP in mg gallic acid equivalents /
g of dry extract was significantly higher (p <0.05) in
ethanolic extracts than in aqueous extracts. Moreover,
EthUAE extract contains a significantly higher TP than
EthMAC extract. Compared to the standardized extract
®
Oligopin
which is purified to be enriched in
polyphenols, C.cornuta ethanolic extracts had lower TP
contents but considering that these are crude extracts,
they can be regarded as rich in polyphenols. On the
other hand, the lowest content in TP was determined in
WUAE extract composed of only 17% of phenolic
compounds which is significantly lower than WMAC
extract. It is clear from this analysis that the extraction
technique and the solvent have a direct influence on
TP content.
In general, the aqueous extracts contain more
flavonoids than ethanolic extracts while ethanolic
extracts are richer in hydroxycinnamic acids (Table 1).
TheW MAC extract has a double Total flav content
®
compared to the standardized extract, Oligopin , while
the total phenols content of WUAE is comparable to
Oligopin. It is interesting to observe that even if WUAE
extract has the lowest TP content, the flavonoids
represent 4.1 ± 3.8 % of polyphenols present in this
extract, which is similar to WMAC extract (4.7 ± 0.5 % of
its TP content) and significantly higher than EthMAC
extract (0.5 ± 0.1% of its TP content). Ultrasounds
seem therefore to favor the extraction of this class of
polyphenols. Indeed, Total flav content in EthUAE extract
is double of EthMAC extract. Phenolic acids contents in
C. cornuta dry crude extracts are much lower than that
®
determined for Oligopin . In the same way, levels of
proanthocyanidins in beaked hazel twig extracts are
®
ten times lower than that of Oligopin . EthMAC extract is
the one which was determined to have the highest
contents in these two classes of polyphenols. One
should keep in mind however, that other classes of
polyphenolic compounds which are not taken into
account in this analysis, may play a key role in
bioactivities of these extracts.
3.2. Trolox Equivalent Antioxidant Capacity
Figure 1 shows that all beaked hazel twig extracts
prepared at the same concentration in TP in the extract
solution demonstrated a TEAC greater than or similar
®
to Oligopin . The highest TEAC was obtained with
WUAE extract, meaning that 1 mg of W UAE extract TP
has a reducing power equivalent to about 14 mg of the
pure antioxidant, Trolox. Thus, for an equivalent
concentration in TP in µg/ mL of extract solutions, C.
Cornuta extracts show quasi identical antioxidant
capacity.
3.3. Radical Scavenging Capacity Against DPPH
The results presented at Figure 2 show that
C.cornuta twig extracts present a high capacity to
74
International Journal of Biotechnology for Wellness Industries, 2012 Vol. 1, No. 1
Royer and Stevanovic
Figure 1: Total Antioxidant Capacity (TAC) of C.cornuta twig extracts taken at the same concentrations in total phenols (250 µg
®
of TP in GAE/mL of extract solution) and compared to Oligopin at 250 µg/mL.
Bars with different superscript letters are significantly different at p <0.05 (ANOVA followed by Duncan's test). The extract with
the letter "a" is the most antioxidant.
scavenge the synthetic radical, DPPH. Indeed,
compared to the standard antioxidants (α-tocopherol,
propyl gallate, ascorbic acid and BHT), commonly used
in food or cosmetics, all extracts exhibit a comparable
or better antiradical activity. The pure antioxidant propyl
gallate is the one which provides the strongest
antiradical activity against DPPH. Among C.cornuta
twig extracts, the highest rate constant was determined
for WMAC extract and thus the WMAC extract has a
capacity to react quickly and scavenge DPPH radical.
This extract has an activity comparable to ascorbic acid
(vitamin C).
3.4. Evaluation of Scavenging Activity Against
Reactive Oxygen and Nitrogen Species (ROS/RNS)
Results presented in Table 2 put in evidence the
chemical antiradical properties of C. cornuta extracts.
However, one should keep in mind that these results
cannot be used to interpret the complexity of the
interactions of antioxidants in vivo. While there is no
ambiguity about the validity of the in vitro data and
these can be used as basis for planning in vivo and
clinical studies, an in vitro assay cannot be
extrapolated to an in vivo situation. Therefore, the
Figure 2: Constant rate (k in s-1) of DPPH scavenging by C. cornuta twig extracts at the concentration of 250 µg in Total
Phenols/mL of extract solution. Extract activities were compared to standard antioxidant at the concentration of 250 µg/mL.
Bars with different superscript letters are significantly different at p <0.05 (ANOVA followed by Duncan's test). The extract with
the letter "a" is the most antioxidant.
Study of Corylus cornuta Twig Extracts
International Journal of Biotechnology for Wellness Industries, 2012 Vol. 1, No. 1
75
Table 2: IC50 Values Determined for C. cornuta Extracts Scavenging Various ROS and RNS
ROS
PRIMARY ROS
IC50 *
OH·
WMAC
850.5 ± 52.0
a
223.8 ± 20.3
828.0 ± 33.5
a
a
WUAE
EthMAC
Oligopin
H2O2
inactive
EthUAE
O ·
c
128 ± 16.8
181.1 ± 20.2
a,b
226.7 ± 33.4
b
6.3±1.5
ROO·
a
11.5±1.5
c
inactive
®
SECONDARY ROS/RNS
2
d
333.2±7.5
a
562.5±133.6
70.7±44.6
a
119.1±9.3
a
1034.6 ± 20.9
347.9 ± 88.3
Curcumin
-
-
492.7±109.4
Quercetin
-
-
295.4±8.9
Ascorbic acid
-
-
-
b
NO·
b
a
249.3±11.2
c
b
377.4±25.0
c
516.6±41.6
1072.8±51.6
c
d
HOCl
66.6±5.5
a,b
n.d
48.9±6.1
a,b
n.d
87.9±21.9
a,b,c
a,b,c
144.3±31.5
d
2083.3±196.3
inactive
inactive
b
471.8±27.5
a
-
16.3±4.7
-
-
-
-
-
864.3±19.1
c
Means with different letters in the same column are significantly different at p<0.05 (ANOVA, followed by Duncan test). *IC50 are expressed in µg TP in GAE eq/mL of
extract solution instead of the conventional µg dry extract/mL solution.
supporting evidence of in vivo antioxidant capacity
should also be provided. These assays do not take into
account the bioavailability, in vivo stability, in vivo
storage patterns by tissues and the reactivity of the
sample matrix in vivo, nor do they reflect the well
documented synergistic effects of antioxidants.
IC50 presented in Table 2 demonstrate that aqueous
extracts are the most effective extracts to scavenge
OH·, O2 ·, ROO· and NO· (p< 0.05). W MAC extract is the
most active of C. cornuta extracts with a capacity to
inhibit 50% of the superoxide radical at a very low
concentration. The analysis of dose-response curves
showed also that activity of the extracts increases in a
dose-dependence manner. At a maximum dose tested
of 500 µg of TP / mL of extract solution, aqueous
extracts exhibited the highest percentage of inhibition
(≈ 90%) of superoxide anion which is statistically
®
comparable to the Oligopin taken at the same
concentration. When the extracts were exposed to
H2O2, they were determined to be significantly more
®
active than Oligopin , WUAE extract being the most
active when considering its low IC50 in TP / mL of
extract solution (p< 0.05). But it should be taken into
account that it corresponds to 730 µg / mL of dry crude
extract. In fact, in terms of crude extract EthMAC would
be more effective because it has a higher TP content
per g of dry extract (Table 1) and would require a lower
dose (≈ 440 µg / mL) of crude extract to reach a level
of phenols permitting the inhibition of 50% of H2O2. C.
cornuta twig extracts were weakly active against OH•.
The two aqueous extracts have lower IC50 than the
®
ethanolic extracts and Oligopin but the dose required
in µg TP/mL of extract solution to inhibit 50% of the
hydroxyl radical OH • is over 500 µg TP /mL of extract
solution, which would correspond to more than 2000 µg
of dry extract/mL. Hydroxyl radical is produced by the
reaction of superoxide radical with hydrogen peroxide.
We have shown that C.cornuta aqueous extracts
strongly inhibit superoxide radical and hydrogen
peroxide (Table 2). So despite the fact that these
extracts are only slightly active against the hydroxyl
radical itself, they are capable of strongly inhibiting its
formation via their high reactivity with both primary
ROS which are involved in hydroxyl radical formation.
When exposed to peroxyl radical, W UAE extract is the
most active but the other extracts are also effective and
®
all extracts exhibit an IC50 lower than that of Oligopin
(p <0.05). Here again, the relative proportions of the
various classes of polyphenols present in WUAE extract
seem to play a role in the activity of this extract
compared to its analogue obtained by maceration. The
study of extract capacity to scavenge NO·
demonstrates that the lowest IC50 is obtained with the
reference, curcumin, a well-known anti-inflammatory
compound [44]. However, the aqueous extracts of C.
cornuta twigs present a comparable activity to that of
this pure compound (p<0.05). Besides, all extracts are
®
significantly more active than Oligopin which has an
IC50> 2000 µg/mL and which can be considered as
inactive against NO•. The two most active extracts
against NO• demonstrate the highest proportion in
flavonoids in their phenolic composition. Test of the
activity of C. cornuta twig extracts against the powerful
oxidant HOCl released during inflammatory processes
was performed in vitro in the presence of natural
antioxidant enzyme system, catalase, an enzyme that
destroys the primary ROS, H2O2 to produce water and
oxygen. Indeed, HOCl is an oxidant enough powerful to
disable this antioxidant enzyme. The capacity of the
extracts to inhibit the deactivation of the enzyme and
so, to scavenge HOCl was evaluated at several
concentrations in µg TP/ mL of the extract solution. The
results presented in Table 2 show that C. cornuta twig
76
International Journal of Biotechnology for Wellness Industries, 2012 Vol. 1, No. 1
extracts were not active or showed very weak capacity
to scavenge HOCl compared to ascorbic acid or
®
Oligopin . Activity was only detected for the two
aqueous extracts which demonstrate inhibition of the
catalase deactivation by HOCl inferior to 50%. WMAC
extract is active in the range of 176.3 ± 15.4 to 863.3 ±
14.8 µg total phenols/ mL of extract solution and WUAE
extract is active in the range of 131.4 ± 12.9 to 668.8 ±
16.7 µg total phenols/ mL of extract solution. In these
ranges of concentrations, both extracts inhibit around
35% of catalase deactivation by the oxidant HOCl.
Overall, the results obtained indicate that
polyphenols contained in WUAE extract are the most
efficient radical scavengers, whereas ethanolic
polyphenolic extracts are quite similar in their behavior
against radical species. The interpretation of the
activities either based on the concentration in total
phenols (such as purified extracts) or on the
concentration in crude extract (containing a mixture of
various kinds of molecules in addition to phenols)
allowed us to identify the ultrasonic water extract as the
one with the broadest spectrum of different classes of
phenols in proportions (% w/w of each class of
phenols/ total phenol content). Thus a purification of
this extract could provide an extract with even greater
activity.
3.5. Capacity of the Extracts to Inhibit Lipidic
Peroxidation
All lipids containing unsaturated fatty acids
regardless of their origin (vegetable oils, fish oils,
animal fats, cell membranes, lipoproteins) are
susceptible to peroxidation. The study of the
mechanisms of lipid peroxidation and its prevention by
antioxidants is experiencing a resurgence of interest
due to the implications of this phenomenon in the areas
of nutrition and health. In food, for both humans and
animals, lipid oxidation is a serious problem for the
food industry that uses more highly unsaturated fatty
acids extremely susceptible to oxidation. The oxidation
of dietary fat leads to qualitative (rancidity, odor) and
nutritional (loss of vitamins and amino acids) alterations
or even toxicity caused by products of lipid peroxidation
(accumulation of peroxides, aldehydes, etc. in food)
[57]. This remains true for cosmetics, themselves rich
in polyunsaturated fatty acids. Peroxidation has been
related to cellular aging and various diseases such as
Parkinson's and Alzheimer's as well as schizophrenia,
atherosclerosis, inflammatory diseases, heart damage
and reperfusion ischemia [58]. The study of the lipid
peroxidation inhibition of linoleic acid (an Omega-3)
Royer and Stevanovic
permits to evaluate the extracts in a system closer to
reality. This test evaluates the influence of extracts on
the oxidative stability of lipids present in our bodies as
well as in manufactured products such as food (for
humans and animals) and cosmetics rich in
polyunsaturated fatty acids.
The activity of the extracts was compared to that of
four standards used as antioxidants: ascorbic acid,
BHT, Trolox and propyl gallate. The results presented
in Table 3 show that W MAC extract with an IC50 of 30.7 ±
53.0 µg / mL total phenols (corresponding to a mass of
crude extract of 94 µg/mL) has an activity significantly
higher than that of the other extracts and all tested
antioxidant references (p<0.05). WUAE is the second
most powerful extract, which inhibits 50% of lipid
peroxidation at a very low concentration. Its activity is
comparable to that of BHT. EthMac extract which
presents an activity comparable to that of vitamin C is
also more active than the ultrasonic extract obtained
with the same solvent. The EthUAE extract is the least
efficient and it is less active than all antioxidant
references.
Table 3: IC50 of C. cornuta Twig Extracts Determined to
Evaluate their Capacity to Inhibit Lipidic
Peroxidation
Extracts
IC50 (µg of total phenols/mL of extract
solution)
WMAC
30.7 ± 53.0
WUAE
96.7 ± 36.7
EthMAC
224.7 ± 81.3
EthUAE
549.5 ± 36.0
Ascorbic acid
244.7 ± 24.8
BHT
a
a,b
b,c
e
c
129.8 ± 15.9
a,b,c
c
Propyl gallate
255.7 ± 18.6
Trolox
457.3 ± 57.3
d
Means with different letters in the same column are significantly different at
p<0.05 (ANOVA, followed by Duncan test).
3.6. Correlations Between Antioxidant/Antiradical
Activities and Polyphenolic Composition
Spearman’s correlation data presented in Table 6
show that the correlation coefficients characterizing the
relationship
between
antioxidant
/
antiradical
efficiencies (calculated as AE = 1/IC50) measured for
each in vitro test and the content of total phenols (TP)
determined in aqueous and ethanolic extracts of
C.cornuta twig extracts, are all significantly (p-value
<0.05) negative, except for TEAC for which no
Study of Corylus cornuta Twig Extracts
International Journal of Biotechnology for Wellness Industries, 2012 Vol. 1, No. 1
significant correlation can be observed. Thus, in
general, more C. cornuta extracts present a high TP
content per gram less they are antioxidant/antiradical.
Several studies have been made concerning the
relationship between the phenolic structure and antioxidant activity [59], but no relationship has been
elucidated because of the many different evaluation
systems used to test for anti-oxidant activity. Several
studies have demonstrated a linear relationship
between anti-oxidant capacity and total phenolics [60,
61]. According to them different phenolic compounds
have different responses in the Folin-Ciocalteau
method. Negative correlation between TEAC and total
phenolic content was found in strawberries [62]. The
authors supposed that this phenomenon could be due
to a high content in ascorbic acid. Kahkonen et al. [63]
found that no significant correlations could be found
between the total phenolic content and antioxidant
activity of various plant extracts and considered that
the antioxidant activity of an extract cannot be
predicted on the basis of its total phenolic content.
Correlation coefficients between hydroxycinnamic acids
(THCA) and antiradical activities were not significant,
except for DPPH scavenging, for which the coefficient
value is significantly negative (p-value <0.05). This
class of polyphenolic compounds seems not to be
involved in the activity of C. corylus twig extracts
against reactive species or lipidic peroxidation. On the
other hand, the results presented in Table 6 indicate
clearly the strong influence of flavonoids on the general
antioxidant/antiradical activities of C.cornuta twig
extracts, including their capacity to inhibit lipid
peroxidation. The only ROS not affected by flavonoids
is HOCl. The various activities are directly proportional
to flavonoid content of the extracts and correlate
significantly (p-value <0.05) with them, except for nitric
oxide for which the correlation is less significant (pvalue <0.1). The most significant correlation has been
found between Total flavonoid content and superoxide
anion scavenging. In the literature, it is well-established
that flavonoids are excellent scavengers of superoxide
anion O2-• [64]. Regarding the correlations between
proanthocyanidin content (PAs) and antioxidant /
antiradical activities, it was found that the capacity of
the extracts to scavenge superoxide anion and DPPH
radical is negatively correlated with PAs (p-value
<0.05) while a low positive correlation is found between
PAs and nitric oxide scavenging.
As the results of the study of the correlations
between the phenolic
composition and
the
antioxidant/antiradical activities of C.cornuta twig
77
extracts indicate, the strong influence of flavonoids has
been determined, while there are no significant effects
of hydroxycinnamic acids or proanthocyanidins.
3.7. Enzyme Modulators
From the general point of view, the ethanolic
extracts are those which demonstrate the highest
capacity to react with enzymes. The first observation in
this sense can be made from analysis of the results
obtained by in vitro test performed to determine
antioxidant activity of C. cornuta twig extracts against
HOCl in presence of catalase (Figure 3). Catalase is an
antioxidant enzyme that is capable of scavenging
oxygen and organic free radicals such as H2O2. It was
interesting to see via this test that the two ethanolic
extracts were acting by “promoting” catalase
deactivation from the very low dose in total phenols
(<50 µg TP/ mL extract solution). This phenomenon
appears at the dose-response curve by a negative
inhibition rate (Figure 3). This behavior could be
explained by complex combination of interactions and
also by solubility restrictions as observed by Aruoma et
al. [45]. Mathematic models applied on the doseresponse curves permitted us to estimate for each
extract the concentration from which a deactivation of
the catalase is observed in presence of these extracts
(Table 4). Results show that aqueous extract
demonstrate this antagonist effect only at very high
dose in TP in the medium. It has already been proved
that high dose of polyphenolic antioxidants can provoke
pro-oxidative mechanisms [65, 66]. Moreover, it is
known that bovine catalase used for this test is easily
deactivated [67]. Like traditional heterogeneous
catalysts, enzymes also suffer from the common failing
of being rather easily and irreversibly poisoned
(saturated), often by reactants (substrates) and
reaction products. It was shown that bovine liver
catalase is deactivated by the direct action of substrate,
the hydrogen peroxide [68]. Indeed, our extracts do
contain intrinsically free H2O2 responsible for the
oxidation mechanisms of phenols (data not shown).
This could explain a promotion of the deactivation of
catalase in presence of certain extracts when HOCl is
added to the system. On the other hand, studies on
maritime pine bark extract (PBE, which corresponds to
®
Oligopin ), which is a mixture of bioflavonoids and
proanthocyanidins, have demonstrated that PBE dosedependently inhibited the activity and changed the
electrophoretic mobility of catalase under certain
conditions. This study also suggested that PBE extract
had an effect on catalase activity by binding to the
enzyme [48]. Further investigations should be
78
International Journal of Biotechnology for Wellness Industries, 2012 Vol. 1, No. 1
Royer and Stevanovic
Figure 3: Inhibition percentages of catalase deactivation by HOCl in presence of C. cornuta twig extracts.
performed on a proper enzymatic assay of catalase in
order to study the mechanism of action of C.cornuta
twig extracts and determine their selectivity to modulate
enzyme activities. Therefore, there could be two
phenomena explaining the reduction of catalase activity
towards HOCl in the presence of phenolic extracts:
contamination of the enzyme by the intrinsically
produced H2O2 and/or
by
binding of
the
proanthocyanidins to the active sites of the enzyme.
C. cornuta twig extracts were also tested against an
enzyme that catalyzes the oxidation of its substrate by
molecular oxygen and hence generates either
intermediate or end product-reactive oxygen species in
the enzymatic reaction. Xanthine oxidase (XO)
catalyzes the generation of superoxide anion and H 2O2
by reduction of O2, while the hypoxanthine and
xanthine are oxidized to uric acid. XO is considered an
important biological source of superoxide radical
involved in the process of oxidative stress [48]. The
activities of the various extracts tested at several
concentrations of total phenols/ mL of extract solutions
were compared with those of the maritime pine bark
®
extract, Oligopin (DRT, Dax, France), quercetin and
curcumin (Sigma Aldrich, St. Louis, MO, USA). The
results are presented in Table 4. All extracts showed
®
an activity against XO higher than Oligopin . EthMAC
and WUAE extracts were the two most active followed by
Table 4: IC50 of C. cornuta Twig Extracts Determined to Evaluate their Capacity to Inhibit the Two Oxidation
Enzymes:Catalase and Xanthine Oxidase
Extracts and references
Catalase deactivation
Xanthine oxidase
IC50 (µg of total phenols/mL of extract solution)
IC50 (µg of total phenols/mL of extract solution)
WMAC
863.3 ± 14.8
c
WUAE
668.8 ± 16.7
b
EthMAC
<50
a
13.1± 7.2
EthUAE
<50
a
73.7 ± 16.4
Oligopin
®
1040.5 ± 30.1
d
e
103.0 ± 9.3
b,c
71.5 ± 29.9
b
a
b,c
1040.5 ± 30.1
Ascorbic acid
>2000
Quercetin
-
0.014 ± 0.006
Curcumin
-
106.2 ± 1.9
Means with different letters in the same column are significantly different at p<0.05 (ANOVA, followed by Duncan test).
d
c
a
Study of Corylus cornuta Twig Extracts
International Journal of Biotechnology for Wellness Industries, 2012 Vol. 1, No. 1
EthUAE extract. Between the two aqueous extracts, it
can be noticed that the phenolic compounds present in
WUAE are significantly more active than those present in
WMAC extract. The activity of C.cornuta twig extracts
increased in a dose-dependence manner. At the
highest dose tested (1000 µg total phenols/mL of
extract solutions), extracts inhibit 100% of the enzyme
®
while Oligopin inhibits 43% of the enzyme activity.
C.cornuta extracts deactivate XO at very low doses in
total phenols. It has been demonstrated that maritime
pine bark extract (PBE) changes also the
electrophoretic mobility of XO and that the inhibition of
this enzyme by this proanthocyanidin-rich extract can
be explained by a phenomenon of complexation of XO
by PBE [48]. Indeed, EthMAC and WUAE extracts are the
richest in proanthocyanidins (Table 1). Spearman’s
correlation data presented in Table 6 show a low
positive correlation between TP content and the
capacity of the extracts to inhibit xanthine oxidase.
Considering the various classes of polyphenols, it
seems that phenolic acids and proanthocyanidins,
more important in ethanolic extracts, are involved in
their anti-enzymatic activities as shown by the
significant and strong positive correlations determined
(p-value <0.05). Our results are in agreement with
other studies. Indeed, it has already been proved that
some hydroxycinnamic acids such as caffeic acid
inhibit xanthine-oxidase [69, 70]. Chan et al. [69] have
pointed out that the molecular structure of the
hydroxycinnamic acid has a very important influence on
xanthine oxidase inhibition. Proanthocyanidin activity
against xanthine oxidase has also been shown in
several studies [71]. There is a high variation in the
79
possible mechanisms for enzyme inhibition. Moini et al.
[48] have demonstrated that proanthocyanidin-rich
extract of maritime pine bark (PBE) selectively inhibits
xanthine oxidase through binding to the enzyme rather
than by the redox activity. On the other hand, we
observed to a lesser extent, a negative correlation
between flavonoids and xanthine-oxidase inhibition. Lin
et al. [72] showed the importance of the structure of the
flavonoids for the mechanism of xanthine oxidase
inhibition, which is related to the binding of these
compounds to the active sites of the enzyme. These
authors have demonstrated that the 3-substituted
hydroxyl benzopyranone ring exhibited weak inhibitory
effect, which could be explained by the destabilization
of polar hydroxyl stretching into the hydrophobic region
of active site of the enzyme and resulted in lowering
the binding affinity. It has also been demonstrated that
the glycosyl substitution at the C6 position of flavonoid
benzopyranone ring also hinders the binding of
inhibitors into the active site of xanthine oxidase. It may
be possible that some flavonoids contained in C.
cornuta twig extracts present an inadequate structure
for interaction with this enzyme. Further investigation
about the structural elucidation of compounds present
in this extract are necessary in order to better
understand the correlations determined in this
research.
3.8. Toxicological Evaluation of Corylus cornuta
Extracts on Normal Human Kératinocytes
Previous studies have suggested that cultured
human keratinocytes may be predictive of irritancy
caused by various surfactants in human subjects [73]
Table 5: Toxic Doses of the Extracts Corresponding to 50% (IC50) of Cell Viability at Three Lines of NHK for C. cornuta
Extracts After 24 and 48 hrs of Exposition of Normal Human Skin Keratinocytes, Determined by MTT and
Neutral Red Methods
Extracts
WMAC
WUAE
EthMAC
EthUAE
MTT (24h)
MTT (48h)
NR (48h)
IC50 (µg/mL)
IC50 (µg/mL)
IC50 (µg/mL)
>1000
734,5
314,4
>1000
605,5
124
>1000
>1000
236,1
895,3
>1000
399,4
>1000
897,5
213,4
>1000
>1000
248,9
905,4
561,8
> 62,5 a
941,6
715,5
> 62,5
952,3
571,1
n.d.
840,4
518,6
> 62,5
730,2
546,1
> 62,5
712,6
507,9
n.d.
80
International Journal of Biotechnology for Wellness Industries, 2012 Vol. 1, No. 1
and the data provided by this model have shown a
good correlation with in vivo skin irritation [74]. The
toxicological properties of the most efficient antioxidant
extracts (W MAC and WUAE) were evaluated on normal
human keratinocytes during 24 and 48 hours by MTT
and after 48h for Neutral Red method through the
determination of the mean toxicity value (IC50).
Toxicological properties of the vehicle used (cell culture
medium containing 1 % ethanol in the case of ethanolic
extracts) was not found to have any significant effect
on toxicity of keratinocytes by MTT method (data not
shown). Problems of solubility of ethanolic extracts in
the medium do not permit the right calculation of their
respective IC50 by Neutral Red Method after 48h
exposition. The results (Table 5) show that in general,
aqueous extracts are the least toxic on NHEK (p< 0.05)
after 48h while ethanolic extracts are the most toxic. It
is interesting to observe the effect of ultrasounds on the
preferential extraction of molecules, since the toxicity of
the extracts has been shown to depend on the solvent
chosen for extraction. Therefore, it seems that after 48
h, the major contribution to the toxicity could be
attributed to the presence of polyphenolic metabolites
as result of interaction with keratinocytes. Indeed, we
Royer and Stevanovic
observed a decrease of the IC50 after 48h exposition
compared to 24h exposition, meaning that C. cornuta
extract phenolic-oxidation products caused by
peroxidase activity present in kératinocytes contribute
to mitochondrial toxicity [39]. Spearman’s correlation
data presented in Table 6 have been calculated only
with MTT assay results on the basis of IC50 obtained
after 48h exposition to NHEK. The results show that
there is a significant negative correlation between MTT
IC50 and TP content (p-value < 0.05), meaning that the
more TP content, the higher is the cytotoxicity of the
extract. No correlations were found with phenolic acids
with PAs neither. On the other hand, a significant
positive correlation was found between flavonoid
content and the extract cytotoxicity. Flavonoids present
in C. cornuta twig aqueous extracts are responsible of
their high antioxidant/antiradical properties and their
low cytotoxicity.
3.9. Active Dose vs Toxic Dose
In order to know how safe the extracts were for
keratinocytes at their antioxidant/antiradical active
concentrations, we calculated the relationship between
Table 6: Spearman’s Correlation Coefficients Obtained Between Antioxidant/Antiradical and Anti-Enzymatic Activities
as well as Cytotoxicity on NHK and the Polyphenolic Composition of C. cornuta Twig Extracts
Extracts activity
TP
THCA
TFlav
PAs
Superoxide
R= -0.75
p-value = 0.007353
R = 0.27
p-value = 0.2330
R= 0.88
p-value = 0.0001922
R = -0.74
p-value = 0.00817
Hydroxyle
R = -0.59
p-value = 0.04884
R = 0.13
p-value = 0.7435
R = 0.77
p-value = 0.02139
R = 0.12
p-value = 0.7756
Peroxyde Hydrogène
-
-
-
-
Peroxyl
R = -0.73
p-value = 0.009052
R = -0.04
p-value = 0.9037
R = 0.67
p-value = 0.02044
R = 0.02
p-value = 0.9562
Nitric oxide
R= -0.67
p-value = 0.02044
R = 0.14
p-value = 0.6672
R = 0.52
p-value = 0.08388
R = 0.46
p-value = 0.03803
Hypochlorous acid
R= -0.89
p-value = 0.03333
R = -0.28
p-value = 0.3331
R = -0.15
p-value = 0.6158
R = 0.15
p-value = 0.6511
Lipidic peroxidation
R = -0.94
p-value < 2.2e-16
R = -0.35
p-value = 0.256
R = 0.61
p-value = 0.003702
R = -0.35
p-value = 0.266
TEAC
R = -0.29
p-value = 0.3543
R = -0.21
p-value = 0.5135
R = 0.53
p-value = 0.0793
R = -0.20
p-value = 0.5281
DPPH Scavenging
R = -0.79
p-value = 0.003617
R = -0.76
p-value = 0.005897
R = 0.86
p-value = 0.0004433
R = -0.77
p-value = 0.005253
Catalase
-
-
-
-
Xanthine oxidase
R= 0.57
p-value = 0.05903
R = 0.76
p-value = 0.005897
R = -0.64
p-value = 0.02795
R = 0.86
p-value = 0.0005971
IC50 MTT assay (48h)
R= -0.77
p-value = 0.003090
R= 0.02
p-value = 0.948
R= 0.61
p-value = 0.03688
R= -0.01
p-value = 0.9653
2
2
Study of Corylus cornuta Twig Extracts
International Journal of Biotechnology for Wellness Industries, 2012 Vol. 1, No. 1
MTT IC50 values and the antioxidant capacity (IC50).
We found that antiradical concentrations of C.cornuta
aqueous extracts for scavenging H2O2 and ROO• were
approximately 2.4-6.2 times lower than cytotoxic
concentrations whereas their antioxidant/antiradical
concentrations for scavenging O2 • and NO• were
approximately 12.6-125.8 times lower than cytotoxic
concentrations. This last estimation pointed out the
strong effect of C.cornuta twig extracts on reactive
species involved in inflammatory processes. On the
contrary, the effective activity of the extracts to
scavenge OH•was obtained at toxic concentration for
human keratinocytes. On the other hand, the
concentrations of aqueous extracts effective for
inhibiting lipidic peroxidation were approximately 8.727.9 times lower than their cytotoxic concentrations.
Considering ethanolic extracts, the concentrations
required for inhibiting xanthine oxidase were
approximately 7.6-43.8 times lower than their cytotoxic
concentrations. These extracts could be therefore good
candidates for the development of therapeutic drugs to
treat gout. These estimations should not be, however,
considered as
completely
predictive of the
effectiveness of extracts in cell systems at
concentrations lower than IC50. Chemical reactivity is
only a part of bioactivity and in cellular context other
factors such as the activation/deactivation of
polyphenolic compounds as a consequence of
metabolism and the interaction with endogenous
antioxidants should be considered.
polyphenols such as flavonoids or proanthocyanidins.
Aqueous extracts were determined to have high
content of total phenols and flavonoids and low toxicity
on NHK. Our results indicate clearly an important role
of flavonoids in radical scavenging, in particular against
superoxide and hydroxyl radicals, as well as in the
inhibition of lipid peroxidation. The water extracts of C.
cornuta twigs were particularly determined to be more
effective than ethanolic extracts against reactive
species involved in inflammatory processes. This last
property should be further investigated in order to
establish the relevance of these results for the
treatment of symptoms claimed by the traditional
remedies based on water extracts of C. cornuta twigs.
In further investigations, the isolation and identification
of compounds present in the aqueous extract will be
performed, along with various studies on the antiinflammatory potential of C. cornuta aqueous extract.
ACKNOWLEDGEMENTS
The authors are thankful to Pure Hazelwood Inc.,
Sherbrooke, QC, for providing vegetal material and
financial support to carry out this research and Dr
Yannick Piriou of DRT (Dérivés résiniques et
terpéniques), Dax, France for generous gift of the fresh
®
Oligopin samples.
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DOI: http://dx.doi.org/10.6000/1927‐3037.2012.01.01.05 Royer and Stevanovic
2001; 145(5): 709-15.
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Accepted on 18-02-2012
Published on 06-04-2012
© 2012 Royer and Stevanovic; Licensee Lifescience Global.
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